The current paradigm for the development of non-viral DNA delivery vectors is to model viral assembly and gene transfer by incorporating, in combinatorial fashion, functional groups that enable particular assembly and transfer steps. Cationic polymers or lipids are used to condense DNA into small virus-like particles. This condensation step is deemed important for several reasons: a) protection of the DNA from inactivation by blood components, b) protection of the DNA from degradation by extracellular nucleases, c) extravascularization of the particle across small openings (fenestrae) in endothelial barriers (for intravascular administration routes), and d) cellular endocytosis. Functional groups are incorporated into synthetic vectors to enhance cell targeting, endosomal escape, and nuclear targeting of the DNA to be delivered. These signals include cell-surface ligands designed to direct the vector to a particular cell type and/or enhance adsorptive or receptor-mediated endocytic uptake of the particle. The vector may also contain molecules designed to enhance release of endocytosed DNA particle into the cell cytoplasm. While the components of a DNA delivery vehicle are known in theory, forming an efficient non-viral delivery vector in practice has been problematic. Cationic polymers or lipids which are good at condensing DNA tend to be toxic or have poor biodistribution. Similarly, compounds which may possess good endosome disruption activity are also frequently toxic.
While cationic polymers and lipids are essential to condense DNA into nanoparticles, their cationic nature limits their wider utility for in vivo applications not only by low gene expression but by toxicity as well. The intravascular route of administration, an attractive approach for wide spread delivery, is particularly plagued by toxicity as well as biodistribution problems. Decreased transfection efficiency in vivo is due in part to the interaction of the polyplexes or lipoplexes with blood components such as serum proteins which inhibit transfection. This effect is usually attributed to the opsonization of the DNA complexes by serum components. Furthermore, intravenously-injected cationic DNA complexes also encounter unintended cell types such as macrophages, monocytes, neutrophils, platelets and erythrocytes, which are important potential mediators of toxicity. Toxic manifestations of systemically-administered cationic DNA complexes can range from red blood cell agglutination to potent inflammatory reaction and elevated serum levels of liver enzymes. Several studies have attempted to avoid such adverse interactions by including polyethyleneglycol (PEG) or proteins such as albumin or transferrin in the DNA complexes. Another method proposed to decrease the charge of a polycation-condensed DNA particle and thus decrease interaction with serum components is to recharge the DNA/polycation complex by addition of a polyanion. Alternating complexes of polycations and polyanions form layered structures when absorbed on macrosurfaces from aqueous solutions. It has been demonstrated that a similar phenomenon takes place on the surface of polycation-condensed DNA particles when they are further complexed with a third-layer polyanion (U.S. Pat. No. 7,396,919, incorporated herein by reference).
The liver is one of the most important target tissues for gene therapy given its central role in metabolism (e.g., lipoprotein metabolism in various hypercholesterolemias) and the secretion of circulating proteins (e.g., clotting factors in hemophilia). At least one hundred different genetic disorders could be at least partially corrected by liver-directed gene therapy. Their cumulative frequency is approximately one percent of all births. In addition, acquired disorders such as chronic hepatitis and cirrhosis are common and could also be treated by polynucleotide-based liver therapies. Gene therapies involving heterotopic gene expression would further enlarge the number of disorders treatable by liver-directed gene transfer. For example, diabetes mellitus could be treated by expressing the insulin gene within hepatocytes whose physiology may enable glucose-regulated insulin secretion. Gene therapy encompassed the purposeful delivery of genetic material to cells for the purpose of treating disease as well as for biomedical investigation or research. Research can be used to study gene function or to facilitate drug discovery or validation.
While viral vectors are the basis of most pre-clinical studies and human clinical trials for delivery of DNA to liver cells, non-viral approaches are continuing to advance. Polyplexes, lipoplexes and lipopolyplexes have all been proposed for delivery vectors to the liver. Most liver non-viral transfer studies have used polyplexes typically containing poly-L-lysine or PEI and ligands for the asialoglycoprotein receptor (ASGPr). Liposomes and lipopolyplexes for liver gene transfer have been reported as well. We are focused on developing DNA nanoparticles that are better at traversing two critical steps: passing through the circulatory system to gain access to hepatocytes and releasing their genetic cargo from the endosomes. The particles may contain ligands to enhance hepatocyte targeting and uptake.